Transcript Slide 1
In Vitro Developmental Pathways Explant - Piece of tissue put into culture -Tissue selection depends on purpose, species, many factors Explants • Pieces of organs – Leaves – Stems – Roots – Cotyledons – Embryos – Other Explants • Specific cell types – Leaf tissue – Embryo – Pollen – Endosperm – Nucellus Callus • Unorganized, growing mass of cells • Dedifferentiation of explant – Loosely arranged thinned walled, outgrowths from explant – No predictable site of organization or differentiation • Auxin + cytokinin • Often can be maintained indefinitely by subculture, but may lose ability to redifferentiate • Compact vs friable • Habituation Three stages of callus culture • Induction: Cells in explant dedifferentiate and begin to divide • Proliferative Stage: Rapid cell division • Differentiation stage (sometimes): organogenesis or embryogenesis Induction © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS Division E. Sutton, UC Davis Callus © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS Differentiation • Organogenesis • Somatic embryogenesis Cell and Suspension Culture • Cell Cultures? • Suspension Cultures Suspension cultures • Can be initiated from any part of the plant. • Usually initiated from friable callus already growing in culture. • Transferred into liquid medium. Agitation • Breakdown of cell aggregates into smaller clumps of cells • Maintains a uniform distribution of cells and cell clumps in the medium • Provides gas exchange Medium • Same as for callus culture? • Gamborg B5 • Conditioning Growth Curve E. Sutton, UC Davis Batch Cultures • A certain number of cells is used to inoculate the culture, in a given volume • Erlenmeyer flask: volume should be about 20% of flask capacity for aeration. • Roller cultures Continuous Culture • Bioreactors • Closed continuous cultures: Remove some of the media and replace with fresh. Continuous removal or periodic. Terminate growth at harvest. Start over. • Open continuous culture: Not only remove some of media, but cells too. Maintain cell density at optimal level. Can be grown for years. Why is it possible to regenerate in vitro? • Totipotency – Initial state – Competence – Determination – Differentiation Only occurs in a few cells in culture. Why? • Pre-determination prior to culture • Newly formed meristems may act as sinks Meristematic centers might actually produce compounds that inhibit neighboring cells. Organogenesis The formation of organs (such as leaves, shoots, roots) on a plant organ, usually of a different kind. Organogenesis • Rule of thumb: Auxin/cytokinin 10:1100:1 induces roots. • 1:10-1:100 induces shoots • Intermediate ratios around 1:1 favor callus growth. Indirect organogenesis Explant → Callus → Meristemoid → Primordium Indirect Organogenesis • Dedifferentiation – Less committed, more plastic developmental state • Induction – Cells become organogenically competent and fully determined for primordia production – Change in culture conditions? • Differentiation © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS Direct Organogenesis Somatic Embryogenesis Parthenocarpy Apomixis In vitro somatic embryogenesis Soybean – Wayne Parrot, UGA Somatic Embryos • Bipolar • Not connected to explant or callus cells by vascular tissue • In most woody plants, tissue must be juvenile or reproductive Indirect Somatic Embryogenesis Induction • Auxins required for induction –Proembryogenic masses form –2,4-D most used –NAA, dicamba also used Development • Auxin must be removed for embryo development • Continued use of auxin inhibits embryogenesis • Stages are similar to those of zygotic embryogenesis – – – – – Globular Heart Torpedo Cotyledonary Germination (conversion) Maturation • Require complete maturation with apical meristem, radical, and cotyledons • Often obtain repetitive embryony • Storage protein production necessary • Often require ABA for complete maturation • ABA often required for normal embryo morphology – Fasciation – Precocious germination Germination • May only obtain 3-5% germination • Sucrose (10%), mannitol (4%) may be required • Drying (desiccation) – ABA levels decrease – Woody plants – Final moisture content 10-40% • Chilling – Decreases ABA levels – Woody plants Rubber tree from somatic embryo CIRAD Factors that Influence SE • • • • Genotype Growth regulators Carbon source Nitrogen Maturation and Germination (Conversion)